Micellar preparation containing sparingly water-soluble anticancer agent and novel block copolymer

ABSTRACT

A novel micellar preparation in which the solubility of a sparingly water-soluble anticancer agent has been heightened and which after intravenous administration, enables a high blood concentration to be maintained. The preparation has high medicinal activity and/or is reduced in side effects. The micellar preparation is formed from a block copolymer represented by the following general formula (1):  
                 
 
[wherein R1 represents hydrogen or C 1-5  alkyl; R2 represents C 1-5  alkylene; R3 represents methylene or ethylene; R4 represents hydrogen or C 1-4  acyl; R5 represents hydroxyl, optionally substituted aryl C 2-8  alkoxyl, substituted C 1-4  alkylamino, or amino having a residue of either an amino acid or a peptide derivative; n is an integer of 5 to 1,000; m is an integer of 2 to 300; and x is an integer of 1 to 300; provided that the proportion of hydroxy in the R5&#39;s is 0 to 99% and x is not larger than m] and a sparingly water-soluble anticancer agent.

TECHNICAL FIELD

The present invention relates to a micellar preparation formed from anovel block copolymer and a sparingly water-soluble anticancer agent, ananticancer agent containing the same as an active ingredient, and theblock copolymer.

BACKGROUND ART

Many important drugs, particularly anticancer agents, are hydrophobiccompounds which are sparingly soluble in water. In order to achievedesired therapeutic effects using such drugs, it is usually requiredthat the drugs be solubilized for administration to patients. Thus, thesolubilization of a sparingly water-soluble anticancer agent representsan important technique for making a formulation thereof for oral orparenteral use, particularly for producing a formulation for use inintravenous administration.

One method for solubilizing a sparingly water-soluble anticancer agentis addition of a surfactant. By way of example, a polyoxyethylene castoroil derivative (Cremophor) is used to solubilize paclitaxel. Othermethods for solubilizing a sparingly water-soluble anticancer agentinclude use of a micelle-forming block copolymer as a carrier for theagent, described, for example, in Japanese Patent Application LayingOpen (KOKAI) Nos. 6-107565, 6-206815, and 11-335267, and the like, andformation of an included paclitaxel-containing micelle using poly(ethylene oxide)-poly (β-benzylaspartate-co-aspartic acid) blockcopolymer, described in Japanese Patent Application Laying Open (KOKAI)No. 2001-226294.

However, the solubilization method using a surfactant shows harmful sideeffects such as hypersensitive reaction due to the surfactant, and alsohas the problem that, because of low stability of the preparation, agentprecipitation occurs when the solution is stored, or allowed to standfor a long period of time.

In addition, intravenous administration of a pharmaceutical preparationusing a block copolymer as a carrier for a sparingly water-solubleanticancer agent, e.g. a taxane anticancer agent, has not achieved thatit maintains a higher concentration of the agent relative toadministration of the agent alone and leads to enhanced pharmacologicaleffects of the agent and reduced side effects thereof.

Thus, there has been a need for a pharmaceutical preparation whichenhances the solubility of a sparingly water-soluble anticancer agent inwater, which maintains an increased concentration of the agent, andyields enhanced pharmacological effects of the agent and reduced sideeffects thereof.

DISCLOSURE OF THE INVENTION

As the result of intensive studies for solving the above-describedproblems, the present inventors have discovered a micellar preparationcomprising a novel block copolymer and a sparingly water-solubleanticancer agent, thereby accomplishing the present invention.

Thus, the present invention relates to:

1) a micellar preparation formed from a block copolymer represented bygeneral formula (1) below

(wherein R1 represents a hydrogen atom or C₁₋₅ alkyl group; R2represents a C₁₋₅ alkylene group; R3 represents methylene or ethylenegroup; R4 represents a hydrogen atom or a C₁₋₄ acyl group; R5 representsa hydroxyl group, an optionally substituted aryl C₂₋₈ alkoxyl group, asubstituted C₁₋₄ alkylamino group, or an amino group having a residue ofa derivative of an amino acid or a peptide; n is an integer of 5 to1,000, m is an integer of 2 to 300, and x is an integer of 1 to 300;provided that the proportion of a hydroxyl group in the R5 is 0 to 99%and x is not larger than m) and a sparingly water-soluble anticanceragent;

2) the micellar preparation described in the above 1) wherein theproportion of hydroxyl group in the R5s of general formula (1) is 0% to90%;

3) the micellar preparation described in the above 1) wherein, ingeneral formula (1), R1 represents a methyl group, R2 represents atrimethylene group, R3 represents a methylene group, R4 represents anacetyl group, and R5 represents an unsubstituted phenyl C₃₋₆ alkoxylgroup; n is an integer of 20 to 500, m is an integer of 10 to 100, and xis an integer of 1 to 100; provided that x is not larger than m;

4) the micellar preparation described in any one of items 1) to 3)wherein the sparingly water-soluble anticancer agent is a taxaneanticancer agent;

5) the micellar preparation described in the above 4) wherein the taxaneanticancer agent is paclitaxel;

6) An anticancer agent containing, as active ingredient, the micellarpreparation described in any one of items 1) to 5);

7) A block copolymer represented by general formula (1) below

(wherein R1 represents a hydrogen atom or C₁₋₅ alkyl group; R2represents a C₁₋₅ alkylene group; R3 represents a methylene or ethylenegroup; R4 represents a hydrogen atom or a C₁₋₄ acyl group; R5 representsa hydroxyl group, an optionally substituted aryl C₂₋₈ alkoxyl group, asubstituted C₁₋₄ alkylamino group, or an amino group having a residue ofa derivative of an amino acid or a peptide; n is an integer of 5 to1,000, m is an integer of 2 to 300, and x is an integer of 1 to 300;provided that the proportion of a hydroxyl group in the R5 is 0 to 99%and x is not larger than m); and

8) the block copolymer described in item 7) wherein, in general formula(1), R1 represents a methyl group, R2 represents a trimethylene group,R3 represents a methylene group, R4 represents an acetyl group, and R5represents an unsubstituted phenyl C₃₋₆ alkoxyl group; n is an integerof 20 to 500, m is an integer of 10 to 100, and x is an integer of 1 to100; provided that x is not larger than m.

BEST MODE FOR CARRYING OUT THE INVENTION

The micellar preparation of the present invention is formed from a blockcopolymer represented by general formula (1) above (wherein R1represents a hydrogen atom or C₁₋₅ alkyl group; R2 represents a C₁₋₅alkylene group; R3 represents a methylene or ethylene group; R4represents a hydrogen atom or a C₁₋₄ acyl group; R5 represents ahydroxyl group, an optionally substituted aryl C₂₋₈ alkoxyl group, asubstituted C₁₋₄ alkylamino group, or an amino group having a residue ofa derivative of an amino acid or a peptide; n is an integer of 5 to1,000, m is an integer of 2 to 300, and x is an integer of 1 to 300;provided that the proportion of a hydroxyl group in the R5 is 0 to 99%and x is not larger than m) and a sparingly water-soluble anticanceragent.

In the block copolymer used for the micellar preparation of theinvention, R1 is a hydrogen atom or a C₁₋₅ alkyl group, preferably aC₁₋₅ alkyl group. Specific examples of the C₁₋₅ alkyl group includemethyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentylgroups, and the like; a methyl group is particularly preferable.

Specific examples of the C₁₋₅ alkylene group in R2 include methylene,ethylene, trimethylene, tetramethylene groups, and the like; ethyleneand trimethylene groups are preferable.

R3 is a methylene or ethylene group, preferably a methylene group.

R4 is a hydrogen atom or a C₁₋₄ acyl group, preferably C₁₋₄ acyl groupsincluding formyl, acetyl, propionyl, butyloyl groups, or the like; anacetyl group is particularly preferable.

In the block copolymer used for the micellar preparation of theinvention, R5 is a hydroxyl group, an optionally substituted aryl C₂₋₈alkoxyl group, a substituted C₁₋₄ alkylamino group, or an amino grouphaving a residue of a derivative of an amino acid or a peptide, and R5smay be the same or different in one molecule. The proportion of hydroxylgroup in the R5s is 0% to 99%, preferably 0% to 90%, more preferably 15%to 85%, most preferably 35% to 80%.

The aryl C₂₋₈ alkoxyl group may be straight-chain or branched C₂₋₈alkoxyl groups to which an aromatic hydrocarbon group such as a phenylor naphtyl group is bound, including, for example, a phenethyloxy,phenylpropoxy, phenylbutoxy, phenylpentyloxy, phenylhexyloxy,phenylheptyloxy, phenyloctyloxy, naphthylethoxy, naphthylpropoxy,naphthylbutoxy, or naphtylpentyloxy group.

The substituent in the optionally substituted aryl C₂₋₈ alkoxyl groupmay be lower alkoxyl groups such as methoxy, ethoxy, isopropoxy,n-butoxy, or t-butoxy, halogen atoms such as fluorine, chlorine, orbromine, nitro group, cyano group, or the like. The number ofsubstituents in the optionally substituted aryl C₂₋₈ alkoxyl group maybe from one to the maximum number for substitution and all substitutedaryl C₂₋₈ alkoxyl group in which all possible positions are substitutedare embraced in the invention; however, the aryl C₂₋₈ alkoxyl group ispreferably unsubstituted.

The optionally substituted aryl C₂₋₈ alkoxyl group may be, preferablyunsubstituted phenyl C₃₋₆ alkoxyl groups including an unsubstitutedphenylpropoxy, unsubstituted phenylbutoxy, unsubstitutedphenylpentyloxy, or unsubstituted phenylhexyloxy group; an unsubstitutedphenylbutoxy group is particularly preferable.

In R5 of the block copolymer represented by general formula (1) used forthe micellar preparation of the invention, the substituted C₁₋₄alkylamino group may be, for example, an optionally substituted arylC₁₋₄ alkylamino group or the like. The aryl C₁₋₄ alkylamino group may bestraight-chain or branched C₁₋₄ alkylamino groups to which an aromatichydrocarbon group such as a phenyl or naphtyl group is bound, including,for example, a benzylamino, phenethylamino, phenylpropylamino,phenylbutylamino, naphthylmethylamino, naphthylethylamino, ornaphthylbutylamino group.

In the optionally substituted aryl C₁₋₄ alkylamino group, thesubstituent may be a lower alkoxyl group such as methoxy, ethoxy,isopropoxy, n-butoxy, or t-butoxy group, a halogen atom such asfluorine, chlorine, or bromine atom, a nitro group, a cyano group, orthe like. The substituted aryl C₁₋₄alkylamino group in which the numberof substituents is from one to the maximum of substitution and allsubstituted aryl C₁₋₄ alkylamino group in which all possible positionsare substituted are embraced in the invention; however, the aryl C₁₋₄alkylamino group is preferably unsubstituted.

Particularly preferred examples of the optionally substituted aryl C₁₋₄alkylamino group are unsubstituted benzylamino, unsubstitutedphenethylamino groups and the like.

R5 in general formula (1) may be an amino group having a residue of aderivative of an amino acid or a peptide. Such an amino group is aprimary amino group comprised in a derivative of an α- or β-amino acidor a peptide in which two or more amino acids are linked through anamide bonding. The derivative of an amino acid or a peptide may be, forexample, that having the main chain carboxylic acid esterified, the sidechain carboxylic acid esterified, or the side chain hydroxyl groupetherified. Specific examples of these include dibenzyl aspartate,β-alanyl-serine benzyl ether benzyl ester (β-alanyl-O-benzyl-L-serinebenzyl ester), and the like.

In the block copolymer represented by general formula (1) used for themicellar preparation of the invention, n is an integer of 5 to 1,000,preferably 20 to 500, particularly 80 to 400; m is an integer of 2 to300, preferably 10 to 100, particularly 15 to 60; x is an integer of 1to 300, preferably 1 to 100, particularly 1 to 60; provided that x isnot larger than m.

A process for preparing the block copolymer represented by generalformula (1) is not particularly restricted, but may involve, forexample, subjecting a compound in which R5 is an optionally substitutedaryl (C2 to C8) alkoxyl or a substituted (C1 to C4) alkylamino group, topartial hydrolysis using acid or alkali, as described in Japanese PatentApplication Laying Open (KOKAI) Nos. 11-335267 and 2001-226294.

Of block copolymers represented by general formula (1), a compoundhaving a group other than a hydroxyl group in R5 may be also obtained bythe dehydration condensation reaction of a compound of general formula(1) in which R5s are all hydroxyl groups with an optionally substitutedaryl C₂₋₈ alcohol, a substituted C₁₋₄ alkylamine, or a derivative of anamino acid or a peptide. The optionally substituted aryl C₂₋₈ alcohol isan alcohol corresponding to the above-described optionally substitutedaryl C₂₋₈ alkoxyl group. The substituted C₁₋₄ alkylamine is an aminecorresponding to the above-described substituted C₁₋₄ alkylamino group.

As the optionally substituted aryl C₂₋₈ alcohol, the substituted C₁₋₄alkylamine, and the derivative of an amino acid or a peptide,commercially available compounds may be used, or compounds prepared by awell-known method for organic synthesis or compounds prepared byapplying and combining well-known organic reactions may also be used.

A process for preparing the compound in which R5s are all hydroxylgroups is not particularly restricted, but a method described e.g. inJapanese Patent Application Laying Open (KOKAI) No. 6-206815 may beused.

The dehydration condensation agent used in the above-describeddehydration condensation reaction may be, for example, acarbodiimide-based dehydration condensation agent including1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl),dicyclohexylcarbodiimide (DCC), or diisopropylcarbodiimide (DIPCI).

The dehydration condensation agent is preferably used in an amount of0.1- to 20-fold moles, particularly 1- to 5-fold moles of the optionallysubstituted C₂₋₈ alcohol, or the substituted C₁₋₄ alkylamine. Here,there may also coexist hydroxysuccinimide (HOSu), 1-hydroxybenzotriazole(HOBt), N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HOBN),4-dimethylaminopyridine (DMAP), diisopropylethylamine, or the like in anamount of 0.01- to 20-fold moles, preferably 0.1- to 10-fold moles ofthe optionally substituted C₂₋₈ alcohol, or the substituted C₁₋₄alkylamine.

The amount of the optionally substituted C₂₋₈ alcohol, or thesubstituted C₁₋₄ alkylamine used is not particularly restricted, but ispreferably 0.1 to 5 equivalents to one equivalent of the carboxyl groupof the compound of general formula (1) in which R5s are all hydroxyl.

The dehydration condensation reaction is preferably performed in asolvent; as the solvent various solvents may be used such as, forexample, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), dioxane,tetrahydrofuran, water, or a mixture thereof, and is not particularlyrestricted. The amount of solvent used is not particularly restricted,but is usually 1 to 500 folds based on the weight of a raw materialcopolymer.

The dehydration condensation reaction is carried out preferably at −10to 60° C. and could be performed for 2 to 48 hours.

Where the block copolymer represented by general formula (1) used forthe micellar preparation of the invention has carboxyl groups, a saltgenerated by the ionic dissociation of part or all thereof is alsoembraced in the invention. The salt may be an alkali metal salt, analkaline earth metal salt, an ammonium salt, or an organic ammoniumsalt, and for example, specifically including a sodium salt, a potassiumsalt, a calcium salt, an ammonium salt, or a triethylammonium salt.

The sparingly water-soluble anticancer agent in the micellar preparationof the invention refers to such an anticancer agent as is substantiallynot dissolved, per se, in the equivalent quantity of water under ambientenvironment such as room temperature or ordinary pressure, or as ispreferentially distributed into a chloroform phase in a solvent systemof equal amounts of water and chloroform. Such anticancer agents mayinclude anthracycline anticancer agents such as adriamycin, taxaneanticancer agents such as paclitaxel and docetaxel, vinca alkaloidanticancer agents such as vincristine, methotrexate, and derivativesthereof; particularly, a taxane anticancer agent, preferably paclitaxel,is included.

The weight ratio of the block copolymer in the micellar preparation ofthe invention to the sparingly water-soluble anticancer agent is 1,000:1to 1:1, preferably 100:1 to 1.5:1, particularly 20:1 to 2:1. However,when the micellar preparation is water-soluble, the agent may becontained in an amount as much as possible.

The micellar preparation of the invention is prepared, for example, bythe following methods. Method a; Inclusion of agent through stirring Thesparingly water-soluble anticancer agent, optionally dissolved in awater-miscible organic solvent, is stirred and mixed with a blockcopolymer aqueous dispersion. In this respect, heating may be carriedout during the stirring and mixing.

Method B; Solvent Volatilization

The sparingly water-soluble anticancer agent in a water-immiscibleorganic solvent is mixed in a block copolymer aqueous dispersion, andthe organic solvent is volatilized with stirring.

Method C; Dialysis

The sparingly water-soluble anticancer agent and the block copolymer aredissolved in a water-miscible organic solvent, and the resultantsolution is dialyzed against a buffer solution and/or water using adialysis membrane.

Method D; Other Method

The sparingly water-soluble anticancer agent and the block copolymer aredissolved in a water-immiscible organic solvent. The resultant solutionis mixed with water and stirred to form an oil-in-water (O/W) emulsion,followed by volatilizing the organic solvent.

For example, a method for preparing micells in Method cis specificallydescribed e.g. in Japanese Patent Application Laying Open (KOKAI) No.6-107565.

Methods b and d involving the volatilization of an organic solvent aredescribed, for example, in Japanese Patent Application Laying Open(KOKAI) Nos. 11-335267 and 2001-226294.

More specifically describing Methods b and d, the water-immiscibleorganic solvent refers to a solvent having the opposite conception todimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile, and thelike which can be substantially freely mixed with water, used forformation of polymer micells e.g. in Japanese Patent Application LayingOpen (KOKAI) No. 11-335267, and is not particularly restricted, but maybe chloroform, methylene chloride, toluene, n-hexane, or a mixturethereof.

The water-immiscible organic solvent is mixed with an aqueous medium,i.e. water (including purified water or ion-exchanged water) or anisotonized or buffered aqueous solution containing saccharide,stabilizer, sodium chloride, buffering agent, or the like. Here, awater-miscible organic solvent or other inorganic salt (e.g. sodiumsulfate) may be contained in a small amount, to the extent that theformation of the O/W emulsion is not adversely affected.

The water-immiscible organic solvent and the aqueous medium aretypically mixed in such a way that the volume ratio is adjusted to1:100, preferably 1:20. As the mixing means may be used a commonly usedmeans for preparing various emulsions, e.g. a mechanical stirrer, ashaking machine, an ultrasonic irradiator, or the like. In this case,operation temperature is not restricted, but is preferably set to therange of about −5° C. to about 40° C. in consideration of temperaturestability of agent, boiling point of solvent, and the like.

Subsequently, the above-described mixing operation is continued in anopen system, or the organic solvent is evaporated (or volatilized) forremoval under reduced pressure with stirring.

The micellar preparation aqueous solution may be subjected to filtrationtreatment for insoluble matter or precipitate directly, or afterultrasonication when the micellar preparation is likely to be associatedor aggregated. The filtration film used is not restricted, but ispreferably a film having a pore size of 0.1 to 1 μm.

The micellar preparation of the invention is stable in aqueous medium,and can provide a higher concentration of the sparingly water-solubleanticancer agent in water compared to the case of not using theadditive. In addition, in order to increase the concentration of thisagent-containing micellar preparation, enrichment through reducedpressure or ultrafiltration, lyophilization, or the like can be made.

In the micellar preparation, the concentration of agent is 0.1 to 50% byweight, preferably 1 to 40% by weight, particularly 5 to 35% by weightbased on the total weight of the agent and the block copolymer;therefore, the amount of the agent may be about 0.01 mg or more,preferably about 1 mg or more, particularly about 2 mg or more for 1 mLof a micellar preparation aqueous solution.

The micellar preparation of the invention may be in the form of acore-shell type micelle in which the structural part of polyethyleneglycol is set to the outside in aqueous medium, and includes thesparingly water-soluble anticancer agent in the hydrophobic portioninside the micell. In the case of the core-shell type micelle, theparticle size can be measured using a commercially available lightscattering particle size measuring device (for example, OtsukaElectronics, Co., Ltd., Model DLS-7000DH); the average particle size is10 to 200 nm, preferably 20 to 100 nm.

An anticancer agent using, as active ingredient, the above anticanceragent-containing micellar preparation is also embraced in the invention.When the micellar preparation is administered as a pharmaceuticalpreparation, the dosage thereof is determined depending on the age,weight and pathology of a patient, the therapeutic purpose, and thelike; however, the therapeutically effective amount thereof isapproximately 50 to 500 mg/body/day. The pharmaceutical preparationadministered is not particularly restricted in so far as it has themicellar preparation dissolved in a pharmaceutically acceptable solvent,and may contain pharmacologically acceptable additives. Micellarpreparations of the invention also include those lyophilized.

In addition, the present invention encompasses the block copolymer usedin the above-described micellar preparation.

EXAMPLES

The present invention is further described, referring to specificpreparation examples. However, the invention is not intended to belimited by these examples.

In these examples, ethanol is abbreviated as EtOH; diisopropyl ether asIPE; 4-dimethylaminopyridine as DMAP; N-hydroxysuccinimide as HOSu; andhigh-performance liquid chromatography as HPLC.

Example 1 Preparing Block Copolymer 6

To 3.56 g of PEG (average molecular weight: 12,000)-pAsp (average degreeof polymerization: 35)—Ac (a compound of general formula (1) where R1represents a methyl group, R2 represents a trimethylene group, R3represents a methylene group, R4 represents an acetyl group, R5represents a hydroxyl group; n is about 272, m is about 35, and x isabout 26; hereinafter abbreviated as PEG-pAsp-Ac) prepared as describedin Japanese Patent Application Laying Open (KOKAI) No. 6-206815, wasadded 70 mL of DMF for dissolving at 35° C., to which DMAP (745 mg),4-phenyl-1-butanol (1.17 mL), and DIPCI (1.19 mL) were then added,followed by reaction for 26 hours. The reaction liquid was addeddropwise to 700 mL of IPE:EtOH (4:1) before filtrating and recoveringthe precipitate, followed by drying under reduced pressure to provide3.19 g of a crude crystal. This crude crystal was dissolved in a 50%acetonitrile aqueous solution, which was then passed through 40 mL of acation exchange resin DOWEX 50w8 (manufactured by Mitsubishi ChemicalCorporation), followed by washing with a 50% water-containingacetonitrile. The eluate was vacuum concentrated and then lyophilized toprovide 3.85 g of block copolymer 6.

The block copolymer 6 (25.2 mg) was dissolved in 2 mL of acetonitrile,to which 2 mL of 0.5N sodium hydroxide aqueous solution was added,followed by stirring at room temperature for 20 minutes. Afterneutralization with 0.5 mL of acetic acid, the fluid volume was adjustedto 5 mL, followed by quantitating free 4-phenyl-1-butanol using HPLC. Asthe result of analysis, the esterically bonded 4-phenyl-1-butanol was44% to the polyaspartic acid.

Example 2 Preparing Block Copolymer 7

By a similar operation to that in Example 1, 3.56 g of block copolymer 7was obtained using 3.59 g of PEG-pAsp-Ac and 0.3 time the amount of4-phenyl-1-butanol (0.36 mL) as that in Example 1.

As the result of analysis following a similar hydrolysis operation tothat in Example 1, the esterically bonded 4-phenyl-1-butanol was 22% tothe polyaspartic acid.

Example 3 Preparing Block Copolymer 8

3.02 g of PEG (average molecular weight: 5,000)-pAsp (average degree ofpolymerization: 30)—Ac (a compound of general formula (1) where R1represents a methyl group, R2 represents a trimethylene group, R3represents a methylene group, R4 represents an acetyl group, R5represents a hydroxyl group; n is about 110, m is about 30, and x isabout 22; hereinafter abbreviated as PEG*-pAsp*—Ac) prepared asdescribed in Japanese Patent Application Laying Open (KOKAI) No.6-206815, was used to perform condensation reaction with4-phenyl-1-butanol (1.45 mL) by a similar operation to that in Example 1to provide 3.05 g of block copolymer 8.

As the result of analysis following a similar hydrolysis operation tothat in Example 1, the esterically bonded 4-phenyl-1-butanol was 50% tothe polyaspartic acid.

Example 4 Preparing Block Copolymer 9

By a similar operation to that in Example 1, 2.74 g of block copolymer 9was obtained using 3.04 g of PEG*-pAsp*—Ac and 0.3 time the amount of4-phenyl-1-butanol (0.44 mL) as that in Example 3.

As the result of analysis following a similar hydrolysis operation tothat in Example 1, the esterically bonded 4-phenyl-1-butanol was 25% tothe polyaspartic acid.

Example 5 Preparing Block Copolymer 10

By a similar operation to that in Example 1, 233 mg of block copolymer10 was obtained using 200 mg of PEG-pAsp-Ac, and 6-phenyl-1-hexanol(80.1 μL) instead of 4-phenyl-1-butanol used in Example 1.

As the result of analysis following a similar hydrolysis operation tothat in Example 1, the esterically bonded 6-phenyl-1-hexanol was 48% tothe polyaspartic acid.

Example 6 Preparing Block Copolymer 13

In 4 mL of DMF was dissolved 200 mg of PEG-pAsp-Ac, to which 49.8 mg ofHOSu, 23.6 μL of benzylamine, and 74.6 μL of DIPCI were then added,followed by reaction at 35° C. for 4 hours. To the reaction liquid wasadded 100 μL of distilled water before stirring for 15 minutes, to which60 mL of IPE EtOH (9:1) was then added dropwise before filtrating andrecovering the precipitate, followed by drying under reduced pressure toprovide 189 mg of block copolymer 13.

The benzylamine remaining in the reaction liquid was quantitated; as theresult of calculation based on that, the amidically bonded benzylaminewas 61% to the polyaspartic acid.

Example 7 Preparing Block Copolymer 15

By a similar operation to that in Example 6, 3.35 g of block copolymer15 was obtained using dibenzyl L-aspartate toluenesulfonate (715 mg) anddiisopropylethylamine (257 μL) instead of benzylamine used in Example 6,and 3.09 g of PEG-pAsp-Ac.

By hydrolysis in similar conditions to those in Example 1, freebenzylalcohol was quantitated for analysis; dibenzyl L-aspartateamidically bonded to block copolymer 15 was 23% to the polyasparticacid.

Example 8 Preparing Block Copolymer 16

By a similar operation to that in Example 6, 1.49 g of block copolymer16 was obtained using β-alanyl-O-benzyl-L-serine benzyl esterhydrochloride (321 mg) preparable by a conventional dipeptide synthesismethod and diisopropylethylamine (142 μL) instead of benzylamine used inExample 6, and PEG-pAsp-Ac (1.51 g).

As the result of analysis using a similar method to that in Example 7,β-alanyl-O-benzyl-L-serine benzyl ester amidically bonded to blockcopolymer 16 was 26% to the polyaspartic acid.

Example 9 Preparing Paclitaxel Micellar Preparation 10

300 mg of block copolymer 6 in Example 1 (a block copolymer with4-phenyl-1-butanol condensed) was weighed into a screw tube bottle, towhich 30 mL of a 40 mg/mL maltose aqueous solution was then added beforestirring to form a dispersion, followed by cooling to 4° C. withstirring. Further, 3 mL of a dichloromethane solution of paclitaxel (30mg/mL) was added before stirring without sealing hermetically in arefrigerator for 16 hours, followed by ultrasonication (130 W, 1secPulse, for 10 minutes). Macrogol 4,000 was added to a concentrationof 20 mg/L for dissolution, followed by filtrating in sterile conditionto provide micellar preparation 10.

The concentration of paclitaxel was 2.8 mg/mL. The average particle sizewas 86.6 nm as determined by a dynamic light scattering photometer(Otsuka Electronics, Co., Ltd., Model DLS-7000DH).

Similarly, the block copolymers described in Examples were used toproduce paclitaxel micellar preparations. The results obtained are shownin Table 1. TABLE 1 Micellar preparations Agent Micellar Blockconcentration Particle size preparation copolymer (mg/mL) (nm) 10 6 2.886.6 11 7 3.1 97.8 12 8 2.8 53.3 13 9 3.0 85.3 18 15 2.0 85.1 19 15 4.520.1 20 16 2.0 28.8

Comparative Example

In accordance with Japanese Patent Application Laying Open (KOKAI) No.6-107565, 10 g of a polyethylene glycol derivative having a methoxygroup on one terminal and an amino group on another terminal andcommercially available β-benzyl L-aspartate N-carboxylic anhydride weredissolved in 80 mL of the mixed solvent of DMF/DMSO (50%/50%) forreaction at 40° C. for 24 hours with shielding the light using aluminumfoil. Subsequently, the reaction solution was added dropwise to 660 mLof the mixed solvent of n-hexane/ethyl acetate (50%/50%) toreprecipitate the polymer. Three rounds of the reprecipitation operationwere conducted, followed by drying under reduced pressure for 24 hoursto provide about 19 g of poly(ethylene oxide; average molecular weight:12,000)-poly(β-benzyl aspartate; average degree of polymerization: 50)block copolymer.

In 100 mL of acetonitrile was dissolved 10 g of the resultant blockcopolymer, to which 22.72 mL of a 0.5 N sodium hydroxide aqueoussolution was then added for hydrolysis reaction at room temperature for10 minutes, The reaction was terminated by adding 3.79 mL of 6 Nhydrochloric acid, followed by transferring the reaction liquid to adialysis membrane (Spectra/Por7, MWCO 3,500) to perform dialysis against3.3 L of ion exchanged water for 9 hours or more (the ion exchangedwater was replaced three times or more). After the end of dialysis,filtration was carried out using a filter paper No. 5B (Kiriyama GlassWorks Co., 4 μm), followed by lyophilization to provide about 9 g ofpoly(ethylene oxide)-poly(β-benzyl aspartate-co-aspartic acid) blockcopolymer, about 50% of which was hydrolyzed.

300 mg of the resultant block copolymer was weighed into a screw tubebottle, to which 30 mL of a 40 mg/mL maltose aqueous solution was addedbefore stirring to make a dispersion, followed by cooling to 4° C. withfurther stirring. 3 mL of a dichloromethane solution of paclitaxel (20mg/mL) was added before stirring without sealing hermetically in arefrigerator for 16 hours, followed by ultrasonication (130 W, 1secPulse, for 10 minutes).

Part of the sample was taken and subjected to particle sizedetermination using a dynamic light scattering photometer (OtsukaElectronics, Co., Ltd., Model DLS-7000DH). The average particle size was107 nm.

Subsequently, Macrogol 4,000 was added to a concentration of 20 mg/mLfor dissolution before filtrating in sterile condition, followed bylyophilization to provide a micellar preparation for ComparativeExample.

Test Example 1 In Vivo Antitumor Effect Against Colon 26

Female CDF1 mice were inoculated subcutaneously at the back thereof withthe mouse colon cancer Colon 26 cells, and, from the point of time whenthe volume of tumor has reached around 100 mm³, micellar preparation 10according to the invention, the micellar composition from ComparativeExample, or paclitaxel was administered through the tail veins of themice for consecutive 5 days to examine effects against advanced cancer.Each agent was used directly, or after dilution with saline before use.The concentration of each agent was expressed in terms of paclitaxel.The antitumor effect of agent was judged on the basis of a percentage ofthe average tumor volume in treated group to the average tumor volume inuntreated group (T/C %) 11 days after agent treatment. The resultsobtained are shown in Table 2. TABLE 2 Antitumor effects against themouse colon cancer Colon 26 Dose Administered agent (mg/kg) T/C (%)Micellar preparation 10 30 13.4 Micellar preparation from 30 42.2Comparative Example Paclitaxel 30 37.5 Paclitaxel 30 40.0

As shown in Table 2, paclitaxel alone exhibited tumor shrinkage rates of37.5% or 40.0% in the 30 mg/kg treated group as compared to untreatedgroup 11 days after treatment; the benzyl ester-type micellarpreparation from Comparative Example exhibited the rate of 42.2%,showing almost the same effect as paclitaxel alone; however, micellarpreparation 10 according to the invention exhibited the rate as low as13.4%, showing a significantly higher antitumor effect.

Test Example 2 Transition of Paclitaxel Concentrations in the Rat Plasma

Micellar preparation 10 from Example 9 was diluted with normal salinesolution to make an aqueous solution having a concentration of 2.5 mg/mLin terms of paclitaxel. Paclitaxel alone was dissolved in ethanol, towhich Cremophor (Sigma) was then added in the same amount as ethanol toprepare in such a way that the concentration of paclitaxel was adjustedto 25 mg/mL followed by dilution to 2.5 mg/mL with normal salinesolution immediately before administration. Micellar preparation 10 orpaclitaxel alone was administered in an amount equivalent to paclitaxel5 mg/kg to male SD rats through their tail veins, followed by takingblood samples from their cervical veins with time. The plasma obtainedby centrifugation was diluted with an appropriate amount of water andsubjected to three rounds of extraction with t-butyl methyl ether. Theether layer was recovered, dried up, and then dissolved in 50 μL ofactonitrile, followed by quantitating paclitaxel using HPLC. The resultsobtained are shown in Table 3. TABLE 3 Paclitaxel concentrations (μg/mL)in the rat plasma Micellar Blood drawing time (min.) preparation 10Paclitaxel 10 66.01 1.05 30 53.68 0.51 60 38.27 0.26 120 30.02 0.10 3607.07 0.035 AUC (0-360) (min · μg/mL) 10236 70.42

As indicated in Table 3, the administration of the micellar preparationof the invention leaded to the maintenance of higher plasmaconcentrations of paclitaxel than that of paclitaxel alone. In the areaunder the blood concentration-time curve (AUC), paclitaxel alone:micellar preparation of the invention was 1: about 150.

Test Example 3 Toxicity Test Against Mouse Extension Reflex (PeripheralNerve Impairment)

Micellar preparation 10 or paclitaxel alone was administered to femaleCDF1 mice through their tail veins for consecutive 5 days to observe theextension reflex of their hind limbs providing an indication ofperipheral nerve impairment due to paclitaxel. Each agent was preparedas described in Test Example 1, and used directly, or after dilutionwith saline before use. The dosages used were expressed in terms ofpaclitaxel. The results obtained are shown in Table 4. TABLE 4 Toxicityagainst mouse extension reflex (Peripheral nerve toxicity) Dose Miceshowing extension Administered agent (mg/kg) reflex disappearanceMicellar preparation 10 30 0/3 Paclitaxel 30 3/3

As indicated in Table 4, paclitaxel alone induced the disappearance ofextension reflex in all mice of the 30 mg/kg treatment group. Incontrast, micellar preparation 10 produced no disappearance of extensionreflex in the 30 mg/kg treatment group. The micellar preparation of theinvention reduced peripheral nerve toxicity as a side effect ofpaclitaxel, compared to paclitaxel alone.

ADVANTAGE OF THE INVENTION

The micellar preparation of the invention enables a necessary amount ofagent, particularly paclitaxel, to be entrapped in micells withoutbinding to a macromolecule, and, when administered, leads to themaintenance of increased blood concentrations of paclitaxel compared tothose for paclitaxel alone. As a result, the preparation may have anenhanced medicinal activity compared to paclitaxel alone, and may alsoreduce the toxicity observed in paclitaxel alone, enabling a usefulpharmaceutical preparation to be provided. In addition, an anticanceragent containing the micellar preparation as active ingredient is alsoprovided.

Further, a block copolymer suitable for forming the micellar preparationwith the above-described effect is also provided. Use of the blockcopolymer has enabled the solubility of a sparingly water-solubleanticancer agent in water to be heightened.

In addition, an aqueous solution of the micellar preparation of theinvention produces, when left at rest at room temperature, noassociation of micells or release of agent from the micellar preparationfor at least several hours, enabling the provision, by the invention, ofa micellar preparation containing a sparingly water-soluble anticanceragent, retained stably in aqueous medium.

1. A micellar preparation formed from a block copolymer represented by general formula (1) below

(wherein R1 represents a hydrogen atom or C₁₋₅ alkyl group; R2 represents a C₁₋₅ alkylene group; R3 represents a methylene or ethylene group; R4 represents a hydrogen atom or a C₁₋₄ acyl group; R5 represents a hydroxyl group, an optionally substituted aryl C₂₋₈ alkoxyl group, a substituted C₁₋₄ alkylamino group, or an amino group having a residue of a derivative of an amino acid or a peptide; n is an integer of 5 to 1,000, m is an integer of 2 to 300, and x is an integer of 1 to 300; provided that the proportion of a hydroxyl group in the R5 is 0 to 99% and x is not larger than m) and a sparingly water-soluble anticancer agent.
 2. The micellar preparation according to claim 1 wherein the proportion of hydroxyl group in the R5s of general formula (1) is 0% to 90%.
 3. The micellar preparation according to claim 1, wherein, in general formula (1), R1 represents a methyl group, R2 represents a trimethylene group, R3 represents a methylene group, R4 represents an acetyl group, and R5 represents an unsubstituted phenyl C₃₋₆ alkoxyl group; n is an integer of 20 to 500, m is an integer of 10 to 100, and x is an integer of 1 to 100; provided that x is not larger than m.
 4. The micellar preparation according to any one of claims 1 to 3, wherein the sparingly water-soluble anticancer agent is a taxane anticancer agent.
 5. The micellar preparation according to claim 4, wherein the taxane anticancer agent is paclitaxel.
 6. An anticancer agent containing, as active ingredient, the micellar preparation according to any one of claims 1 to
 5. 7. A block copolymer represented by general formula (1) below

(wherein R1 represents a hydrogen atom or C₁₋₅ alkyl group; R2 represents a C₁₋₅ alkylene group; R3 represents a methylene or ethylene group; R4 represents a hydrogen atom or a C₁₋₄ acyl group; R5 represents a hydroxyl group, an optionally substituted aryl C₂₋₈ alkoxyl group, a substituted C₁₋₄ alkylamino group, or an amino group having a residue of a derivative of an amino acid or a peptide; n is an integer of 5 to 1,000, m is an integer of 2 to 300, and x is an integer of 1 to 300; provided that the proportion of a hydroxyl group in the R5 is 0 to 99% and x is not larger than m).
 8. The block copolymer according to claim 7, wherein, in general formula (1), R1 represents a methyl group, R2 represents a trimethylene group, R3 represents a methylene group, R4 represents an acetyl group, and R5 represents an unsubstituted phenyl C₃₋₆ alkoxyl group; n is an integer of 20 to 500, m is an integer of 10 to 100, and x is an integer of 1 to 100; provided that x is not larger than m. 